Abstract

Stability of single-chain Fvs (scFvs) can be improved by mutagenesis
followed by phage display selection where the unstable variants are
first inactivated by, for example, denaturing treatment. Here we
describe a modified strategy for the selection of stabilized antibody
fragments by phage display, based on denaturation under reducing
conditions. This strategy was applied to an anti-thyroid-stimulating
hormone (TSH) scFv fragment which refolded remarkably during the
selection if denaturation was carried out in conventionally used
non-reducing conditions. Refolding was, however, efficiently prevented
by combining denaturation with reduction of the intra-domain disulfide
bridges, which created favourable conditions for selection of clones
with improved stability. Using this strategy, scFv mutants with 8–9 °C
improved thermal stability and 0.8–0.9 M improved stability for
guanidinium chloride were found after 4–5 enrichment cycles. The most
stable mutants selected contained either LysH66Arg or AsnH52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.

title = "Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions",

abstract = "Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8–9 °C improved thermal stability and 0.8–0.9 M improved stability for guanidinium chloride were found after 4–5 enrichment cycles. The most stable mutants selected contained either LysH66Arg or AsnH52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.",

N2 - Stability of single-chain Fvs (scFvs) can be improved by mutagenesis
followed by phage display selection where the unstable variants are
first inactivated by, for example, denaturing treatment. Here we
describe a modified strategy for the selection of stabilized antibody
fragments by phage display, based on denaturation under reducing
conditions. This strategy was applied to an anti-thyroid-stimulating
hormone (TSH) scFv fragment which refolded remarkably during the
selection if denaturation was carried out in conventionally used
non-reducing conditions. Refolding was, however, efficiently prevented
by combining denaturation with reduction of the intra-domain disulfide
bridges, which created favourable conditions for selection of clones
with improved stability. Using this strategy, scFv mutants with 8–9 °C
improved thermal stability and 0.8–0.9 M improved stability for
guanidinium chloride were found after 4–5 enrichment cycles. The most
stable mutants selected contained either LysH66Arg or AsnH52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.

AB - Stability of single-chain Fvs (scFvs) can be improved by mutagenesis
followed by phage display selection where the unstable variants are
first inactivated by, for example, denaturing treatment. Here we
describe a modified strategy for the selection of stabilized antibody
fragments by phage display, based on denaturation under reducing
conditions. This strategy was applied to an anti-thyroid-stimulating
hormone (TSH) scFv fragment which refolded remarkably during the
selection if denaturation was carried out in conventionally used
non-reducing conditions. Refolding was, however, efficiently prevented
by combining denaturation with reduction of the intra-domain disulfide
bridges, which created favourable conditions for selection of clones
with improved stability. Using this strategy, scFv mutants with 8–9 °C
improved thermal stability and 0.8–0.9 M improved stability for
guanidinium chloride were found after 4–5 enrichment cycles. The most
stable mutants selected contained either LysH66Arg or AsnH52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.